An increase of angiogenesis has been shown in idiopatic myelofibrosis with myeloid metaplasia (MMM) by microvessel density count method but evaluation of circulating angiogenic factors is still incomplete. In 31 patients affected by MMM and in 12 healthy subjects we evaluated the serum levels of VEGF (vascular endothelial growth factor) and correlated VEGF with clinical and laboratory features of disease. We found that MMM patients had circulating VEGF concentrations much higher than controls (median 1208 ng/ml vs 138 ng/ml, P < 0.0001). No correlation was found between VEGF and Hb, WBC, PLT, LDH, creatinine, bone marrow cellularity, fibrosis, splenomegaly, hepatomegaly, and therapy. However, in the subgroup of patients with a normal or low VEGF concentration, a direct correlation between VEGF and platelet count (r = 0.90, P = 0.002) was detected. Moreover, patients with a platelet count <300 × 109/l had VEGF serum levels lower than patients with a higher PLT count (median VEGF 864 vs 1557 pg/ml, P = 0.001). In six patients and in eight controls we also had the opportunity to measure VEGF in the plasma and we calculated that VEGF concentration was much higher in platelet-rich than in platelet-poor plasma and that platetets of MMM patients contained four times more VEGF than those of healthy controls. These results indicate that VEGF is overproduced in MMM, thus confirming an increased angiogenic activity. Platelets are probably a major source of VEGF in MMM but not the only one.
In the last years several reports have highlighted the importance of angiogenesis in neoplastic development not only for solid tumors but also for hematological malignancies.1,2 It is now well known that angiogenesis is an important step in the pathophisiology of both myelo- and lymphoproliferative disease.3,4 In addition, the evaluation of angiogenesis may be of clinical interest and may add some important prognostic information to the predictive value of the currently used staging systems.5 In general, an increased angiogenic activity encompasses for an unfavorable outcome and, in some diseases, the evaluation of this process seems to be an independent prognostic factor.6 Therefore, it is necessary to have a simple and reliable tool for measuring the angiogenic activity in cancer patients not only for a correct assessment of prognosis but also for a more precise evaluation of the response to treatment. Although the microvessel density count is an accepted way of measuring angiogenesis,7 this method suffers from a lack of objectivity and it is not suitable for frequent measurements. Alternatively, the assessment of circulating angiogenic factor concentrations seems to be a simple method of quantifying angiogenesis in cancer patients. Many circulating angiogenic factors have been identified8 but so far the role and contribution of each of them in tumoral angiogenesis is not clear. VEGF (vascular endothelial growth factor) seems to be one of the most important mediators of both normal and tumoral angiogenesis1 and the good preclinical results obtained by blocking VEGF or its receptors indicate that this factor may have a leading position in the hierarchy of angiogenesis mediators.9 Indeed, evaluation of circulating levels of VEGF have shown to be of prognostic value in many solid tumors1,2,10 and in hematological malignancies such as non-Hodgkin lymphomas,11 acute myeloid and lymphoid leukemias,6,12,13 chronic myeloid and lymphoid leukemias13 and myelodysplastic syndromes.14 In addition, it has been demonstrated that VEGF measurement correlates with microvessel density count.15
Idiopathic myelofibrosis with myeloid metaplasia (MMM) is a disease in which angiogenesis is certainly increased,16,17 but so far its role in the pathogenesis of the disease is still uncertain.18 Recent studies have confirmed that in the bone marrow of MMM patients there is an increase of microvessel density that is correlated to the number of cells that stained for VEGF17 and patients with the higher angiogenic activity had the worst prognosis.19 In addition, several studies have reported that patients affected by MMM have increased circulating levels of TGF-β (trasforming growth factor-beta) and bFGF (basic fibroblast growth factor) that, beside other activities, are strong inducers of angiogenesis.20,21,22
On this basis, we have evaluated the circulating serum concentrations of VEGF in a cohort of patients affected by MMM and we have found that most of them had levels much higher than control subjects, thus confirming the increase of angiogenesis in MMM.
Patients and methods
Samples were obtained from 31 consecutive patients affected by idiopathic myelofibrosis with myeloid metaplasia. Mean age was 64 years (median 68, range 34–75) and median duration of disease 38 months (range 0–168). Twenty-four had been previously treated and 12 of them were on treatment with hydroxyurea at the time of the study. Their clinical features are listed in Table 1. Bone marrow cellularity and fibrosis were empirically indicated by a pathologist who evaluated bone marrow biopsies. Control samples were obtained from 12 healthy volunteers aged between 30 and 78 years (mean 46).
After informed consent, serum samples were collected and centrifuged at 1000 g for 10 min within 30 min from collection. In six patients (Nos 3, 7, 8, 14, 18 and 20) and in eight controls, plasma samples (collected using EDTA as an anticoagulant) were also available. Two plasma samples for each individual were centrifuged at 100 and 300 g for 10 min in order to obtain both platelet-rich and platelet-poor plasma. Serum and plasma were aliquoted and stored at −20°C until VEGF evaluation. Before VEGF measurement, platelet-rich plasma were repeatly freezed and thawed in order to lyse platelets.
VEGF (Quantikine; R&D Systems, Minneapolis, MN, USA) concentrations were determined in serum and plasma samples by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. Briefly, this assay employs the quantitative sandwich enzyme immunoassay technique with monoclonal antibodies, specific for VEGF pre-coated on to a microplate. Standard controls and samples (100 μl of plasma) were pipetted into the wells in duplicate. After growth factor binding and washing, an enzyme-linked antibody specific for VEGF was added to each well. After thorough washing, a substrate solution was added to the wells and color developed in proportion to the amount of growth factor bound in the first step. Optical density of each well was determined by a microplate reader at 450 nm. Blank was subtracted both from the standard controls and samples. A standard curve was created by plotting the logarithm of the mean absorbance of each standard vs the logarithm of the soluble factor concentration. Concentrations are reported as picograms per milliliter. Normal ranges of VEGF were 62–707 pg/ml for serum and 0–115 pg/ml for plasma respectively.
Correlation between two parameters was evaluated by the Spearman rank correlation analysis. Nonparametric statistical tests (Mann–Whitney or Wilcoxon) were used for comparison between groups. All P values were two-tailed.
Median serum VEGF concentration in the 31 MMM patients was 1208 ng/ml (range (74–4000). This value was significantly higher than our controls (median 138 pg/ml, with a range from 47 to 990) (P < 0.0001) (Figure 1). Of note, 24/31 MMM patients had VEGF levels higher than the upper limit indicated by the manufacturer of the ELISA assay (707 pg/ml). In six patients, VEGF measurement was subsequently repeated in another sample collected after 1 or 2 months and the concordance for VEGF concentration was between 70 and 93%. In addition, although mean age of MMM was higher than controls, no correlation between VEGF and age was found in both MMM patients (r = 0.04) and normal volunteers (r = 0.13). Although patients not receiving treatment at the time of the study had VEGF levels higher than patients receiving hydroxyurea, the difference was not statistically significant (median 1636 pg/ml for untreated vs 869 pg/ml for treated patients, P = 0.2).
No correlation was found between VEGF serum levels and Hb, WBC count, PLT count, LDH, creatinine, BM cellularity, fibrosis, splenomegaly, hepatomegaly, and therapy. However, in the subgroup of patients with a VEGF serum level within the normal range (⩽717 pg/ml), a strong correlation was found between VEGF and platelet count (r = 0.90, P = 0.002). Accordingly, patients with PLT count <300 × 109/l had VEGF serum levels lower than patients with higher PLT count (median VEGF 864 vs 1557 pg/ml, P = 0.001) and the lowest VEGF levels were seen in patients with thrombocytopenia (Table 1). These data indicate that there is a direct correlation between VEGF and platelets but it is limited to the normal or low values of both VEGF and PLT.
To further investigate this finding, in six MMM patients (Nos 3, 7, 8, 14, 18 and 20) and in eight controls, we had the opportunity to measure VEGF levels in plasma and we found that PLT-rich plasma contained higher VEGF concentrations than PLT-poor plasma. Median PLT-rich/poor plasma ratio was 6.5 (range 1.5–86) for patients and 1.28 (range 0.96–12.7) for controls. However, VEGF concentration in PLT-rich plasma was always lower than serum level both in each of the studied patients and in controls. In patients, median VEGF concentration was 553 pg/ml (range 91–2000) for PLT-rich and 57 pg/ml (range 13–507) for PLT-poor plasma. In control subjects, median VEGF concentration was 48 pg/ml (range 13–242) for PLT-rich and 19 pg/ml (range 10–57) for PLT-poor plasma.
In the same patients and in controls we calculated the ratio between VEGF concentration in PLT-rich plasma and PLT count. This ratio gives a rough estimation of how much VEGF is contained in the platelets23,24 and we found that platelets of MMM patients contained four times more VEGF than those of healthy controls: median 0.99 pg per 106 platelets (range 0.6–3.1) vs 0.25 pg per 106 platelets (range 0.09–0.8) for MMM patients and controls respectively (Figure 2). Taken together, these data indicate that platelets may be a major source of VEGF both in MMM patients and in normal subjects and that platelets from patients contain a higher amount of VEGF. However, these data cannot exclude that the increased levels of VEGF in PLT-rich plasma may be due to an abnormal release from platelets. In addition, in MMM both platelets and megakaryocytes may be larger than normal and actually in a quarter of MMM patients the automated counter did not provide the mean platelet volumes probably due to a wide distribution of platelet size. Therefore, it is possible that MMM platelets contain more VEGF just because of their bigger size. However, this finding by itself cannot fully justify the 10 times higher VEGF serum concentration of MMM patients as compared to healthy controls.
This study shows that patients affected by MMM have high circulating VEGF levels. Since VEGF is one of the most important soluble mediators of angiogenesis1 and its levels correlate with microvessel density count,15,17 this finding confirms the increase of angiogenic activity already demonstrated in this disease.16,17,19
However, in MMM, it is not clear which cells produce this high amount of VEGF. In many patients with several cancers, it has been shown that platelets contain high levels of VEGF23,25,26,27,28 and, in our study, we have found that platelets from MMM patients contain and/or release more VEGF than normal controls. Therefore, it is likely that in MMM, as in other cancers, platelets are an important source of circulating VEGF and act as a carrier of this factor. In addition, it has been demonstrated that megakaryocytes can produce VEGF26,29,30 and it is well known that they are increased in MMM bone marrow with alteration in their function and morphology.18 The importance of megakaryocytes as sources of cytokines involved in MMM fibrosis has been largely demonstrated18 and, in this perspective, the production of VEGF further highlights the role of megakaryocytes as first actors in the scenario of MMM pathogenesis. However, other cells must be involved in the production of the very high level of VEGF found in MMM patients. In our study, in fact, a linear correlation between platelets and VEGF was found only in both low–normal platelet count and low–normal VEGF levels. In addition, the four-fold difference in platelet-contained VEGF cannot explain by itself the 10 times difference in circulating VEGF concentrations between MMM patients and healthy subjects. Finally, levels of VEGF measured in the serum of both patients and normal control were always higher than in platelet-rich plasma even after platelet lysis. Therefore, other cells, also involved in clot formation, may contain and transport VEGF. It has been shown that white blood cells from patients affected by solid tumors contain much more VEGF than normal control.23 In our MMM patients no correlation was found between WBC count and VEGF but different white blood cell fractions were not investigated in our study. Bone marrow stromal cells are also good producers of VEGF31 and their involvement is highly probable in a disease such as MMM where activation of fibroblasts is a main feature.
In our study, VEGF levels did not correlate with any of the clinical or laboratory features of the disease. Therefore, our observation does not clarify the role of VEGF in the pathogenesis of MMM and the biological and clinical meaning of these results remains speculative. One speculation could be based on the fact that pluripotent hemopoietic stem cells express receptor for VEGF.32 Since one typical feature of MMM is the presence of a high number of circulating hemopoietic precursors in the peripheral blood,33 it is possible that the massive amount of circulating VEGF in MMM could have an effect on the excessive production and/or migration of marrow clonogenic cells in MMM. Studies are in progress in our laboratory to test this hypothesis.
The finding of increased angiogenic activity in MMM is in line with the most recent literature demonstrating a role for angiogenesis, not only in the development of disease but also as a prognostic indicator.19 These observations may provide information for a novel strategy of treatment for a disease in which the therapeutic armamentarium is quite poor.18,33 Indeed, recent preliminary reports indicate a good activity of thalidomide, a drug with antiangiogenic properties, in a small cohort of patients affected by MMM.34 In this perspective, we have observed that VEGF measurement in the serum or plasma of MMM patients is an easy and reliable method and it can be used as a valuable tool for measuring the activity of angiogenesis.
Ferrara N, Davis-Smyth T . Biology of vascular endothelial growth factor Endocr Rev 1997 18: 4–25
Kraft A, Weindel K, Ochs A, Marth C, Zmija J, Schumacher P, Unger C, Marmè D, Gastl G . Vascular endothelial growth factor in the sera and effusions of patients with malignant and nonmalignant disease Cancer 1999 85: 178–187
Ratajczak MZ, Ratajczak J, Machalinski B, Majka M, Marlicz W, Carter A, Pietrzkowski Z, Gewirtz AM . Role of vascular endothelial growth factor (VEGF) and placenta-derived growth factor (P1GF) in regulating human haemopoietic cell growth Br J Haematol 1998 103: 969–979
Vacca A, Ribatti D, Presta M, Monischetti M, Iurlaro M, Ria R, Albini A, Bussolino F, Dammacco F . Bone marrow neovascularization, plasma cell angiogenic potential, and matrix metalloproteinase-2 secretion parallel progression of human multiple myeloma Blood 1999 93: 3064–3073
Weidner N, Folkman J . Tumor vascularity as a prognostic factor in cancer. In: De Vita VT, Hellman S, Rosenberg SA (eds) Important Advances in Oncology Lippincott-Raven: Philadelphia 1996 167–190
Aguayo A, Estey E, Kantarjian H, Mansouri T, Gidel C, Keating M, Giles F, Estrov Z, Barlogie B, Albitar M . Cellular vascular endothelial growth factor is a predictor of outcome in patients with acute myeloid leukemia Blood 1999 94: 3717–3721
Vermeulen PB, Gasparini G, Fox SB, Toi M, Martin L, McCulloch P, Pezzella F, Viale G, Weidner S, Harris AL, Dirix LY . Quantification of angiogenesis in solid human tumors: an international consensus on the methodology and criteria of evaluation Eur J Cancer 1996 32A: 2474–2484
Risau W . Mechanisms of angiogenesis Nature 1997 386: 671–674
Kim J . Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vitro Nature 1993 362: 841–844
Dirix LY, Vermeulen PB, Pawinski A, Prove A, Benoy I, De Pooter C, Martin M, Van Oosterom AT . Elevated levels of the angiogenic cytokines basic fibroblastic growth factor and vascular endothelia growth factor in sera of cancer patients Br J Cancer 1997 76: 238–243
Salven P, Teerenhovi L, Joensuu H . A high pretreatment serum vascular endothelial growth factor concentration is associated with poor outcome in non-Hodgkin's lymphoma Blood 1997 90: 3167–3172
Perez-Atayde AR, Sallan SE, Tedrow U, Connors S, Allred E, Folkman J . Spectrum of tumor angiogenesis in the bone marrow of children with acute lymphoblastic leukemia Am J Pathol 1997 150: 815–821
Aguayo A, Kantarjian H, Estey E, Cortes J, Beran M, O'Brien S, Keating M, Freireich E, Thomas D, Rogers A, Albitar M . Plasma levels of VEGF and bFGF in various leukemias and their correlation with angiogenesis Blood 1999 94 (Suppl. 1): 503a, (Abstr. 2252)
Bellamy WT, Richter L, Frutiger Y, Sirjani D, Glinsmann-Gibson B, Grogan TM, List AF . Vascular endothelial growth factor (VEGF) is an autocrine promoter of ALIP and leukemia progenitor formation in myelodysplastic syndromes (MDS) Blood 1999 94 (Suppl. 1): 389a, (Abstr. 1727)
Mattern J, Koomagi R, Volm M . Association of vascular endothelial growth factor expression with intratumoral microvessel density and tumor cell proliferation in human epidermoid lung carcinoma Br J Cancer 1996 73: 931–934
Thiele J, Rompcik V, Wagner S, Fisher R . Vascular architecture and collagen type IV in primary myelofibrosis and polycythaemia vera: an immunomorphometric study on trephine biopsies of the bone marrow Br J Haematol 1992 80: 227–234
Lundberg LG, Lerner R, Sundelin P, Rogers R, Folkman J, Palmblad J . Bone marrow in polycythemia vera, chronic myelocytic leukemia, and myelofibrosis has an increased vascularity Am J Pathol 2000 157: 15–19
Tefferi A . Myelofibrosis with myeloid metaplasia N Engl J Med 2000 342: 1255–1265
Mesa RA, Hanson CA, Rajkumar SV, Schroeder GS, Tefferi A . Evaluation and clinical correlations of microvessel density in myelofibrosis with myeloid metaplasia Blood 2000 96: 3374–3380
Dalley A, Smith JM, Reilly JT, MacNeil S . Investigation of calmodulin and basic fibroblast growth factor (bFGF) in idiopathic myelofibrosis: evidence for a role of extracellular calodulin in fibroblast proliferation Br J Haematol 1996 93: 856–862
Le Bousse-Kerdiles M-C, Chevillard S, Charpentier A, Romquin N, Clay D, Smadja-Joffe F, Praloran V, Dupriez B, Demory J-L, Jasmin C, Martirè M-C . Differential expression of transforming growth factor-β, basic fibroblast growth factor, and their receptors in CD34+ hematopoietic progenitor cells from patients with myelofibrosis and myeloid metaplasia Blood 1996 88: 4534–4546
Martirè M-C, Le Bousse-Kerdiles M-C, Romquin N, Chevillard S, Praloran V, Demory J-L, Dupriez B . Elevated levels of basic fibroblast grwoth factor in megakaryocyts and platelets from patients with idiopathic myeofibrosis Br J Haematol 1997 97: 441–448
Salven P, Oprana A, Joensuu H . Leukocyte and platelets of patients with cancer contain high levels of vascular endothelial growth factor Clin Cancer Res 1999 5: 487–491
Wynendaele W, Derua R, Hoylaerts MF, Pawinski A, Waelkens E, de Bruijn EA, Paridaens R, Merlevede W, van Oosterom AT . Vascular endothelial growth factor measured in platelet poor plasma allows optimal separation between cancer patients and volunteers: a key to study an angiogenic marker in vivo? Ann Oncol 1999 10: 965–971
Goldberg MA, Schneider TJ . Similarities between the oxygen-sensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin J Biol Chem 1994 269: 4355–4361
Banks RE, Forbes MA, Kinsey SE, Stanley A, Ingham E, Walters C, Selby PJ . Release of the angiogenic cytokine vascular endothelial growth factor (VEGF) from platelets: significance for VEGF measurements and cancer biology Br J Cancer 1998 76: 956–964
Salgado R, Vermeulen PB, Benoy I, Weytjiens R, Huget P, Van Mark E, Dirix LY . Platelet number and interleukin-6 correlate with VEGF but not with bFGF serum levels of advanced cancer patients Br J Cancer 1999 80: 892–897
Verheulen HMW, Hoekman K, Luykx-de Bakker S, Eekman CA, Folman CC, Broxtermann HJ, Pinedo HM . Platelet: transporter of vascular endothelial growth factor Clin Cancer Res 1997 3: 2187–2190
Katoh O, Tauchi H, Kawaishi K, Kimura A, Satow Y . Expression of the vascular endothelial growth factor (VEGF) receptor gene, KDR, in hematopoietic cells and inhibitory effet of VEGF on apoptotic cell death caused by ionizing radiation Cancer Res 1995 55: 5687–5692
Mohle R, Green D, Moore MA, Nachman RL, Rafii S . Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets Proc Nat Acad Sci USA 1997 94: 663–668
Huang Y-Q, Li J-J, Hu L, Karpatkin S . Thrombin induces vascular endothelial cell growth factor (VEGF) in human tumor cells and fibroblasts Blood 1999 94 (Suppl. 1): 12a, (Abstr. 40)
Ziegler BL, Valtieri M, Almeida Porada G, De Maria R, Muller R, Masella B, Gabbianelli M, Casella I, Pelosi E, Bock T, Zanjani ED, Peschle C . KDR receptor: a key marker defining hematopoietic stem cells Science 1999 285: 1553–1558
Barosi G . Myelofibrosis with myeloid metaplasia: diagnostic definition and prognostic classification for clinical studies and treatment guidelines J Clin Oncol 1999 17: 2954–2970
Thomas DA, Aguayo A, Giles FJ, Albitar M, O'Brien S, Cortes J, Faderl S, Bivins C, Zeldis J, Keating MJ, Barlogie B, Kantarjian MJ . Thalidomide anti-angiogenesis therapy (RX) in Philadelphia (Ph)-negative myeloproliferative disorders (MPD) and myelofibrosis (MF) Blood 1999 94 (Suppl. 1): 702a, (Abstr. 3102)
This study was supported in part by FON.CA.NE.SA. (Fondazione Catanese per lo Studio e la Cura delle Malattie Neoplastiche del Sangue).
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Raimondo, F., Azzaro, M., Palumbo, G. et al. Elevated vascular endothelial growth factor (VEGF) serum levels in idiopathic myelofibrosis. Leukemia 15, 976–980 (2001). https://doi.org/10.1038/sj.leu.2402124
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